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Synaptic vesicle (SV) recycling enables ongoing transmitter release, even during prolonged activity. SV membrane and proteins are retrieved by ultrafast endocytosis and new SVs are formed from synaptic endosomes (large vesicles—LVs). Many proteins contribute to SV recycling, e.g., endophilin, synaptojanin, dynamin and clathrin, while the site of action of these proteins (at the plasma membrane (PM) vs. at the endosomal membrane) is only partially understood. Here, we investigated the roles of endophilin A (UNC-57), endophilin-related protein (ERP-1, homologous to human endophilin B1) and of clathrin, in SV recycling at the cholinergic neuromuscular junction (NMJ) of C. elegans. erp-1 mutants exhibited reduced transmission and a progressive reduction in optogenetically evoked muscle contraction, indicative of impaired SV recycling. This was confirmed by electrophysiology, where particularly endophilin A (UNC-57), but also endophilin B (ERP-1) mutants exhibited reduced transmission. By optogenetic and electrophysiological analysis, phenotypes in the unc-57; erp-1 double mutant are largely dominated by the unc-57 mutation, arguing for partially redundant functions of endophilins A and B, but also hinting at a back-up mechanism for neuronal endocytosis. By electron microscopy (EM), we observed that unc-57 and erp-1; unc-57 double mutants showed increased numbers of synaptic endosomes of large size, assigning a role for both proteins at the endosome, because endosomal disintegration into new SVs, but not formation of endosomes were hampered. Accordingly, only low amounts of SVs were present. Also erp-1 mutants show reduced SV numbers (but no increase in LVs), thus ERP-1 contributes to SV formation. We analyzed temperature-sensitive mutants of clathrin heavy chain (chc-1), as well as erp-1; chc-1 and unc-57; chc-1 double mutants. SV recycling phenotypes were obvious from optogenetic stimulation experiments. By EM, chc-1 mutants showed formation of numerous and large endosomes, arguing that clathrin, as shown for mammalian synapses, acts at the endosome in formation of new SVs. Without endophilins, clathrin formed endosomes at the PM, while endophilins A and B compensated for the loss of clathrin at the PM, under conditions of high SV turnover.
The capacity of convalescent and vaccine-elicited sera and monoclonal antibodies (mAb) to neutralize SARS-CoV-2 variants is currently of high relevance to assess the protection against infections. We performed a cell culture-based neutralization assay focusing on authentic SARS-CoV-2 variants B.1.617.1 (Kappa), B.1.617.2 (Delta), B.1.427/B.1.429 (Epsilon), all harboring the spike substitution L452R. We found that authentic SARS-CoV-2 variants harboring L452R had reduced susceptibility to convalescent and vaccine-elicited sera and mAbs. Compared to B.1, Kappa and Delta showed a reduced neutralization by convalescent sera by a factor of 8.00 and 5.33, respectively, which constitutes a 2-fold greater reduction when compared to Epsilon. BNT2b2 and mRNA1273 vaccine-elicited sera were less effective against Kappa, Delta, and Epsilon compared to B.1. No difference was observed between Kappa and Delta towards vaccine-elicited sera, whereas convalescent sera were 1.51-fold less effective against Delta, respectively. Both B.1.617 variants Kappa (+E484Q) and Delta (+T478K) were less susceptible to either casirivimab or imdevimab. In conclusion, in contrast to the parallel circulating Kappa variant, the neutralization efficiency of convalescent and vaccine-elicited sera against Delta was moderately reduced. Delta was resistant to imdevimab, which, however, might be circumvented by combination therapy with casirivimab together.
Influenza A (H1N1) 2009 : impact on Frankfurt in due consideration of health care and public health
(2010)
Background: In April 2009 a novel influenza A H1N1/2009 virus was identified in Mexico and in the United States which quickly spread around the world. Most of the countries established infection surveillance systems in order to track the number of (laboratory-confirmed) H1N1 cases, hospitalizations and deaths. Methods: The impact of the emergence of the novel pandemic (H1N1) 2009 virus on Frankfurt was statistically evaluated by the Health Protection Authority, City of Frankfurt am Main. Vaccination rates of the health care workers (HCWs) of the University Hospital Frankfurt were measured by the Occupational Health Service. Results: Although the virulence of pandemic (H1N1) 2009 seems to be comparable with seasonal influenza, a major patient load and wave of hospital admissions occurred in the summer of 2009. Even though the 2009 vaccination rate of the University Hospital Frankfurt (seasonal influenza [40.5%], swine flu [36.3%]) is better than the average annual uptake of influenza vaccine in the German health care system (approximately 22% for seasonal and 15% for swine flu), vaccination levels remain insufficient. However, physicians were significantly (p < 0.001) more likely to have been vaccinated against swine flu and seasonal influenza than nurses. Conclusions: The outbreak of the pandemic (H1N1) 2009 in April 2009 provided a major challenge to health services around the world. Nosocomial transmission of H1N1/2009 has been documented. Present experience should be used to improve pandemic preparedness plans and vaccination programs ought to target as many HCWs as possible.
Synaptic vesicles (SVs) undergo a cycle of biogenesis and membrane fusion to release transmitter, followed by recycling. How exocytosis and endocytosis are coupled is intensively investigated. We describe an all-optical method for identification of neurotransmission genes that can directly distinguish SV recycling factors in C. elegans, by motoneuron photostimulation and muscular RCaMP Ca2+ imaging. We verified our approach on mutants affecting synaptic transmission. Mutation of genes affecting SV recycling (unc-26 synaptojanin, unc-41 stonin, unc-57 endophilin, itsn-1 intersectin, snt-1 synaptotagmin) showed a distinct ‘signature’ of muscle Ca2+ dynamics, induced by cholinergic motoneuron photostimulation, i.e. faster rise, and earlier decrease of the signal, reflecting increased synaptic fatigue during ongoing photostimulation. To facilitate high throughput, we measured (3–5 times) ~1000 nematodes for each gene. We explored if this method enables RNAi screening for SV recycling genes. Previous screens for synaptic function genes, based on behavioral or pharmacological assays, allowed no distinction of the stage of the SV cycle in which a protein might act. We generated a strain enabling RNAi specifically only in cholinergic neurons, thus resulting in healthier animals and avoiding lethal phenotypes resulting from knockdown elsewhere. RNAi of control genes resulted in Ca2+ measurements that were consistent with results obtained in the respective genomic mutants, albeit to a weaker extent in most cases, and could further be confirmed by opto-electrophysiological measurements for mutants of some of the genes, including synaptojanin. We screened 95 genes that were previously implicated in cholinergic transmission, and several controls. We identified genes that clustered together with known SV recycling genes, exhibiting a similar signature of their Ca2+ dynamics. Five of these genes (C27B7.7, erp-1, inx-8, inx-10, spp-10) were further assessed in respective genomic mutants; however, while all showed electrophysiological phenotypes indicative of reduced cholinergic transmission, no obvious SV recycling phenotypes could be uncovered for these genes.
Optogenetic manipulation of neuronal activity through excitatory and inhibitory opsins has become an indispensable experimental strategy in neuroscience research. For many applications bidirectional control of neuronal activity allowing both excitation and inhibition of the same neurons in a single experiment is desired. This requires low spectral overlap between the excitatory and inhibitory opsin, matched photocurrent amplitudes and a fixed expression ratio. Moreover, independent activation of two distinct neuronal populations with different optogenetic actuators is still challenging due to blue-light sensitivity of all opsins. Here we report BiPOLES, an optogenetic tool for potent neuronal excitation and inhibition with light of two different wavelengths. BiPOLES enables sensitive, reliable dual-color neuronal spiking and silencing with single- or two-photon excitation, optical tuning of the membrane voltage, and independent optogenetic control of two neuronal populations using a second, blue-light sensitive opsin. The utility of BiPOLES is demonstrated in worms, flies, mice and ferrets.
Locomotion circuits developed in simple animals, and circuit motifs further evolved in higher animals. To understand locomotion circuit motifs, they must be characterized in many models. The nematode Caenorhabditis elegans possesses one of the best-studied circuits for undulatory movement. Yet, for 1/6th of the cholinergic motor neurons (MNs), the AS MNs, functional information is unavailable. Ventral nerve cord (VNC) MNs coordinate undulations, in small circuits of complementary neurons innervating opposing muscles. AS MNs differ, as they innervate muscles and other MNs asymmetrically, without complementary partners. We characterized AS MNs by optogenetic, behavioral and imaging analyses. They generate asymmetric muscle activation, enabling navigation, and contribute to coordination of dorso-ventral undulation as well as anterio-posterior bending wave propagation. AS MN activity correlated with forward and backward locomotion, and they functionally connect to premotor interneurons (PINs) for both locomotion regimes. Electrical feedback from AS MNs via gap junctions may affect only backward PINs.
Manipulation of neuronal or muscular activity by optogenetics or other stimuli can be directly linked to the analysis of Caenorhabditis elegans (C. elegans) body length. Thus, WormRuler was developed as an open-source video analysis toolbox that offers video processing and data analysis in one application. Utilizing this novel tool, the super red-shifted channelrhodopsin variant, ChrimsonSA, was characterized in C. elegans. Expression and activation of ChrimsonSA in GABAergic motor neurons results in their depolarization and therefore elongation of body length, the extent of which providing information about the strength of neuronal transmission.
Cardiac arrhythmias are often associated with mutations in ion channels or other proteins. To enable drug development for distinct arrhythmias, model systems are required that allow implementing patient-specific mutations. We assessed a muscular pump in Caenorhabditis elegans. The pharynx utilizes homologues of most of the ion channels, pumps and transporters defining human cardiac physiology. To yield precise rhythmicity, we optically paced the pharynx using channelrhodopsin-2. We assessed pharynx pumping by extracellular recordings (electropharyngeograms--EPGs), and by a novel video-microscopy based method we developed, which allows analyzing multiple animals simultaneously. Mutations in the L-type VGCC (voltage-gated Ca(2+)-channel) EGL-19 caused prolonged pump duration, as found for analogous mutations in the Cav1.2 channel, associated with long QT syndrome. egl-19 mutations affected ability to pump at high frequency and induced arrhythmicity. The pharyngeal neurons did not influence these effects. We tested whether drugs could ameliorate arrhythmia in the optogenetically paced pharynx. The dihydropyridine analog Nemadipine A prolonged pump duration in wild type, and reduced or prolonged pump duration of distinct egl-19 alleles, thus indicating allele-specific effects. In sum, our model may allow screening of drug candidates affecting specific VGCCs mutations, and permit to better understand the effects of distinct mutations on a macroscopic level.
Channelrhodopsin-2 (ChR2) is widely used for rapid photodepolarization of neurons, yet, as it requires high-intensity blue light for activation, it is not suited for long-term in vivo applications, e.g. for manipulations of behavior, or photoactivation of neurons during development. We used “slow” ChR2 variants with mutations in the C128 residue, that exhibit delayed off-kinetics and increased light sensitivity in Caenorhabditis elegans. Following a 1 s light pulse, we could photodepolarize neurons and muscles for minutes (and with repeated brief stimulation, up to days) with low-intensity light. Photoactivation of ChR2(C128S) in command interneurons elicited long-lasting alterations in locomotion. Finally, we could optically induce profound changes in animal development: Long-term photoactivation of ASJ neurons, which regulate larval growth, bypassed the constitutive entry into the “dauer” larval state in daf-11 mutants. These lack a guanylyl cyclase, which possibly renders ASJ neurons hyperpolarized. Furthermore, photostimulated ASJ neurons could acutely trigger dauer-exit. Thus, slow ChR2s can be employed to long-term photoactivate behavior and to trigger alternative animal development.
Optogenetic approaches using light-activated proteins like Channelrhodopsin-2 (ChR2) enable investigating the function of populations of neurons in live Caenorhabditis elegans (and other) animals, as ChR2 expression can be targeted to these cells using specific promoters. Sub-populations of these neurons, or even single cells, can be further addressed by restricting the illumination to the cell of interest. However, this is technically demanding, particularly in free moving animals. Thus, it would be helpful if expression of ChR2 could be restricted to single neurons or neuron pairs, as even wide-field illumination would photostimulate only this particular cell. To this end we adopted the use of Cre or FLP recombinases and conditional ChR2 expression at the intersection of two promoter expression domains, i.e. in the cell of interest only. Success of this method depends on precise knowledge of the individual promoters' expression patterns and on relative expression levels of recombinase and ChR2. A bicistronic expression cassette with GFP helps to identify the correct expression pattern. Here we show specific expression in the AVA reverse command neurons and the aversive polymodal sensory ASH neurons. This approach shall enable to generate strains for optogenetic manipulation of each of the 302 C. elegans neurons. This may eventually allow to model the C. elegans nervous system in its entirety, based on functional data for each neuron.